Amyotrophic lateral sclerosis (ALS) is a prevalent progressive neurodegenerative disease involving the loss of functional neuromuscular junctions (NMJ) and motor neurons (MN), and subsequent paralysis. No effective cures for ALS exist and current therapies only marginally impact disease course. This is because at the time of diagnosis, the disease has typically progressed into the final stages which impedes effective treatment. Therefore, treatment of ALS with novel therapies is an unmet need. Since veterans of the Persian Gulf War are twice as likely to develop ALS, discovering successful therapies is especially significant to our veteran population. Adipose-derived stem/stromal cells (ASC) have been shown to repair and rescue tissues from ischemia and other pathological conditions due to their beneficial secretions including anti-apoptotic, anti-inflammatory, and pro-angiogenic growth factors and cytokines. Recent studies have demonstrated the therapeutic value of conditioned medium isolated from ASC cultures (ASC-CM), and Phase I clinical trials utilizing ASC-CM to treat acute lung injury and specific cardiovascular pathologies are also ongoing. In this context, we have identified a B6SJL hybrid mSOD1G93A mouse model of ALS in which disease progression is similar to that observed in ALS patients. Evidence shows ASC-CM administration in this ALS mouse model after symptom onset improves lumbar spinal cord MN survival and prolongs lifespan. Moreover, early ASC-CM treatment (at disease onset) preserves intact NMJ. Therefore, early ALS treatment using ASC-CM may simultaneously target peripheral and central components of ALS. Using the mSOD1G93A ALS mouse model, we will also administer local muscular injection of a novel bioengineered ASC-CM-embedded polyethylene glycol (PEG) hydrogel that allows slow release of growth/protective factors at the site of neuromuscular disconnection to investigate the specific site(s) of ASC-CM action. We will also assess the overall efficacy for clinical translation of ASC-CM through optimized amelioration of disease progression. Three specific Aims have been designed to elaborate on this areas of investigation. Aim 1: Determine ASC-CM dose response on early NMJ loss, and assess neuromuscular and immune responses to long-term ASC-CM therapy in mSOD1G93A mice. A) First, an experiment will be performed to determine the optimal therapeutic dose of ASC-CM required to preserve intact NMJs. B) The optimal dose of ASC-CM from (A) will be administered from PD35-PD47 and NMJs and MN will be quantified weekly from PD56-91 and compared with C) the optimal dose of ASC-CM administered from PD35-continuously. Aim 2: Determine the therapeutic efficacy of early long-term continuous ASC-CM administration in mSOD1G93A mice. Using the optimal ASC-CM dose established in Aim 1, symptom onset, disease progression rate, and lifespan will be assessed and histologic analyses performed. To assess symptom onset and progression an array of functional assessments will be performed including electromyography (EMG), grip strength, locomotor activity, etc. Aim 3: Determine whether site of disease onset (NMJ) is the site of ASC-CM protective/reparative benefits using a novel slow-release biomaterial applied directly to muscle tissue in mSOD1G93A mice. Following in vitro assessment of ASC-CM release from PEG hydrogel, ASC-CM-PEG hydrogel will be injected into the medial gastrocnemius muscle and NMJ, MN loss and immune response over time will be examined. The results of this aim will shed light on the ASC-CM site of action on NMJ preservation and is a new clinically applicable invention for localized ASC-CM therapy.